Method and means for performing assays



y 30, 1961 J. F. PAGANO EI'AL 2,986,497

METHOD AND MEANS FOR PERFORMING ASSAYS Filed Feb. 26, 1959 5 l4 INVENTORS.

JOSEPH F. PAGANO y EUGENE V. ADAMS ATTORNEY METHOD AND mans non PERFORMING ASSAYS This invention relates to .a device for assaying microorganisms.

Prior to this invention, circular paper discs impregnated with various chemotherapeutic agents have been used 'to determine the sensitivity spectrum of microorganisms. Thus, .for example, to determine the anti- "biotic spectrum of a microoran'gism, .the organism was grown in an agar-containing culture medium and a num- 'beriof discs, each impregnated with a ditierentantibiotic,

where placed upon the medium. After a predetermined time, the sensitivity of the microorganism to a given antibiotic'was determined by the'zone of inhibition (clear area) around the discs.

The disadvantages of such procedure are manifest. Thus, the testing'methodrequiredthe careful placing of a plurality of discs on the nutrient medium; atime-consuming tedious procedure requiring the recordation of the contents and-position ofeach-disc. Furtherfore, there is .a common'misconception in the art that'the comparative activity ofthe impregnating substances is relatedto the :diameter of the one of inhibition orgrowth stimulation. -In other words, the larger the zones, the greater the \potencyof-a given substance, wherein-fact quite often the area of the zone depended on non-pertinent physical characteristics of the impregnating substances, such as their relative diffusion rates through the agar medium.

It is an object of this invention, therefore, to provide a unitary simple device for assaying microorganisms, which is eflicient to use.

It is another object of this invention to provide a device for assaying microorganisms which cannot be misread or misinterpreted.

It is still another object of this invention to provide a device for assaying microorganisms which is easily prepraed commercially.

These objects are achieved by the device of this invention, which comprises a sheet of bibulous material having a plurality of spaced apertures therein, the portion of material peripheral to at least one aperture being impregnated with a microorganism-aiiecting substance. [By microorganism-afiecting substance is meant a substance, such as an antibiotic, phage or assimilable carbon or nitrogen containing compound, which affects the growth pattern of at least one microorganism. This efiect may be one of inhibition or enhancement and includes bacteriocidal, bacteriostatic, and nutrient substances] The device of this invention and its method of preparation and use are illustrated in the accompanying drawing, wherein:

Figure 1 is a top view of one embodiment of this invention;

Figure 2 is a perspective view of the device of this invention in place on a petri dish;

Figure 3 is a perspective view of a means for preparing the devices of this invention showing the impregnation of the devices with microorangism-atfecting substances; and

2,986,497 Patented May 30, 1961 2 Figure 4 is a. sectional "view taken through 4-4 of Figure 3. p

Considering Figure l'of the'drawing, the device for assaying microorganisms of this invention comprises a sheet of bibulous material 1, such as porous paper (e.g. filter paper) having a plurality of spaced apertures 2, 3, 4, 5,

and 6 therein. A portion of sheet l peripheral to a least one of the apertures is impregnated with a microorganism-afiecting substance. In the device shown in Figure 1, portions of the sheet peripheral to apertures 3, 4, 5 and 6 are "impregnated with. difierent v microorganism-affecting substances, such as difierent antibiotics. ..To aid in identifying which aperture is surrounded by a given material, the sheet is preferably provided with indica, as indicated by A, B, C, D, andE, adjacent to the respective apertures. Although the sheet and apertures shown in Figure 1 are circular in outline, these shapes are merely illustrative and rectangular. or other shape sheets may be used. Furthermore, sheets containing apertures of difier- .ent areas Imay also be used.

:Figure 2 shows the devices of thisinvention in use. When so used, a petri dish 7 or other .usual container is prepared with the usual agar-containingnutrient medium 8 and inoculated with the microorganism to be tested. Sheet 1 is then placed on top of the nutrient medium 8 and after apredetermined timethe medium beneath apertures 2, 3, 4, 5 and 6 are observed. .In this time, the microorganism-affectingsubstances surrounding the apertures 'has diffused into .the area of the nutrient medium adjacent thereto including the area .under the. aperture. The eflzect of these substances on the microorganism being tested can then be determined by observing the growth pattern of the microorganismin the area under the aperture.

"Thus, for example, to determine the mode .of treatment of a pathogenic microorganism, .a nutrient medium 8 in petri dish 7 isinoculated with the microorganism and the device of this invention is placed thereon. By way of illustration, the sheet 1 may contain an antifungal antibiotic, such as nystatin or amphotericin,, on the portion peripheral to aperture 3; a broad-spectrum antibiotic, such as tetracycline or chlortetracycline, on the portion peripheral to aperture 4; and narrower spectrum antibiotics, such as streptomycin and penicillin on the portions peripheral to apertures 5 and 6, respectively. The port-ion peripheral to aperture 2 can remain untreated to act as a control. After a given time, the nutrient medium 8 is observed through the apertures. If a microorganism is present, it will be so indicated by a growth (cloudiness) in the nutrient medium under aperture 2. Assuming that a microorganism is present and is an antibiotic-sensitive fungus, such as Candida albicans, its nature will be shown by the lack of growth (clear area) under aperture 3, and growth (cloudiness) under apertures 2, 4, 5 and 6. In a similar manner, the sensitivity to antibiotics of a Grampositive or Gram-negative bacteria can be determined.

Aside from the above use, the device of this invention can also be used to indicate the presence of a specific phage sensitive strain of microorganism by employing a sheet containing one or more apertures, the portions peripheral to each aperture containing a different phage. Furthermore, to indicate the growth pattern of a new microorganism, the microorganism can be grown in an agar medium deficient in a source of carbon and a sheet containing any number of potential sources of assimilable carbon can be placed thereon. Those carbon sources which will support the growth of the microorganism will then be shown by areas of growth (cloudiness) under the respective apertures.

The devices of this invention are readily prepared in sizable quantities by using the apparatus disclosed in Figures 3 and 4. A stock of sheets 1 are aligned so that the apertures therein are in register. Bottom plate 9 is then positioned under the stack and top place 10 is positioned on top of the stack. Threaded bolts 11 are then passed upwards through apertures; 12.,in bottornplate 9 and apertures 13 in top plate 10. The bolts are gprovided with heads 14 of sufficient size to prevent passage of the entire bolts through bottom plate. 9. *Wing nuts .15 are then screwed onto the free endsoi the bolts thereby forcing the plates 9 and 10 together and compressing the stack of sheets 1 therebetween. The plates areqso constructed and the apertures 12 and 13 therein so placed that the bolts pass outside the stack of sheets 1. Top plate 10 is provided with apertures 2', 3', 4', S and'fi' in alignment with theapertures in sheets 1 and bottom plate 9 is preferably provided with' indents, s'u'ch as'indents 4" and also in alignment with'the apertures in the .top :platef, The apertures in top plate 10, and sheets 3. The device of claim 1 wherein the microorganismattecting substances are antibiotics.

4. The device of claim 1 wherein the microorganismalfecting substances are phages.

5. The device of claim 1 wherein the microorganismaffecting substances are sources of assimilable carbon.

6. A device for assaying microorganisms, which comprisesa circular sheet of porous paper having a plurality of spaced circular apertures therein, the portions of paper 1 and the indents bottorn platefl9 thereby provide'a series of wells.

To prepare the devices of this invention, oneor more of the wells are filled with a solution of the desired microorganism-aifecting substances. After a time sulficient for absorption of some of the solution in the area of each sheet 1 peripheral to the well, excess solution is removed within the scope of the appended claims.

What is claimed is:

1. A device for assaying microorganisms which com- 35 prises a sheet of bibulous material having a plurality of spaced apertures, the portions of material peripheral to at least two apertures being impregnated with different microorganism-aflecting substances.

2. The device of claim 1 wherein the bibulous material is porous paper.

'either by inverting the entire assembly or preferably affecting substances are different antibiotics.

peripheral to at least two apertures. being impregnated with different microorganism-infecting substances.

7. The device of claim 6 wherein the microorganismaiiecting substances are antibiotics.

8. A method of assaying'rnicroorganisms,which comprises growing a microorganism on a nutrient medium upon which is positioned a sheet of bibulous material having a plurality of spaced'apertures therein, the portions of material peripheral-to at least two apertures andhaving been impregnated with difierent microorganism-Mime ing substances, and measuring the growth of themicroorganism on the nutrient medium beneath saidIaper'tures.

'9. A method of assaying microorganisms, which coniprises growing a microorganism on a nutrient medium upon which is positioned a sheet of porous paper having a'plurality-of spaced apertures therein, at least two portions of the paper'peripheral to different apertures having been impregnated with diiierent microorganismaiteeting substances, and comparing the extent of growth of the microorganism on the nutrient medium beneath said apertures. j

10. The method of claim 9 wherein the microorganism- References Cited in the file of this patent UNITED STATES PATENTS 742,824 Becker Nov. 3, 1903 1,026,549 Almond May 14, 1912 2,771,399 Savage Nov. 20, 1956 2,787,581 Scherr Apr. 2, 1957 2,791,518 Stokes et al. May 7, 1957 

8. A METHOD OF ASSAYING MICROORGANISMS, WHICH COMPRISES GROWING A MICROORGANISM ON A NUTRIENT MEDIUM UPON WHICH IS POSITIONED A SHEET OF BIBULOUS MATERIAL HAVING A PLURALITY OF SPACED APERTURES THEREIN, THE PORTIONS OF MATERIAL PERIPHERAL TO AT LEAST TWO APERTURES AND HAV- 